This seeks to delineate the role of central cholinergic neurons in the regulation of gonadal steroid hormone levels in rats. Changes in the choline acetyltransferase (ChAT) activity of discrete brain areas in response to the manipulation of circulating levels of gonadal hormones have been used to identify those areas which may participate in the feedback actions of gonadal steroids. In adult male rats, castration elevated ChAT activity in the medial preoptic nucleus and posterior medial amygdala. Testosterone treatment lowered ChAT activity in the nucleus tractus diagonalis. In female rats ovariectomized as adults, estradiol treatment followed 48 hr later by progesterone reduced ChAT activity in the caudal nucleus tractus diagnoalis and in the periventricular nucleus and increased ChAT activity in the supraoptic nucleus. Progesterone alone also reduced ChAT activity in the caudal nucleus tractus diagnolis. These brain areas are among those known to be targets for testicular and ovarian hormones, and may represent sites where circulating gonadal steroids initiate neuroendocrine feedback actions through cholinergic mediators.